CASE REPORT: A case of unusual severe HDFN due to anti-D alloimmunisation in undiagnosed RhD negative primigravida Malay woman is reported here. This case illustrates the possibility of an anamnestic response from previous unknown sensitisation event or the development of anti-D in mid trimester. The newborn expired due to hydrops fetalis and severe anaemia. Antenatally, the mother was identified as RhD positive and thus there was no antenatal antibody screening, antepartum anti-D prophylaxis or close fetal monitoring for HDFN.
DISCUSSION: The thorough antenatal ABO and RhD blood grouping with antibody screening is mandatory as part of prevention and early detection of HDFN especially due to anti-D alloimmunisation. Improper management of RhD negative women might lead to severe HDFN including in primigravida.
MATERIALS AND METHODS: One hundred and twenty primary school children were included. They were divided into caries and caries-free groups. Unstimulated whole saliva was collected from each participant using spitting method. The salivary elements were measured using an Atomic Absorption Spectrophotometer. Descriptive statistics, bivariate and Pearson's correlation analysis were performed.
RESULTS: Salivary Cu and Zn levels were significantly higher in children with dental caries compared to those caries-free (p < 0.05). Moreover, these elements had a positive correlation with dental caries (Cu: r=0.698, p<0.001; Zn: r=0.181, p<0.05). No significant variations in Mn and Fe were observed between caries and caries-free group (p>0.05). Additionally, there were significant differences in salivary Zn and Fe among different age groups (p<0.05) and highly significant differences in salivary Cu, Mn and Fe among different ethnic groups (p<0.001). However, all elements exhibited no significant differences between males and females.
CONCLUSION: The salivary Cu and Zn levels showed significant differences between caries and caries-free groups. The findings also revealed significant variations in the levels of salivary Cu, Mn and Fe among different ethnic groups and salivary Zn and Fe among different age groups.
METHODS: The enzyme was purified in two steps using affinity and size exclusion chromatography. Enzyme assays were performed using the malachite green assay and enzymatic product was identified using gas chromatography-mass spectrometry (GC-MS) analysis. Sequence analysis of PmSTS was performed using multiple sequence alignment (MSA) against plant sesquiterpene synthase sequences. The homology model of PmSTS was generated using I-TASSER server.
RESULTS: Our findings suggest that the recombinant PmSTS is mainly expressed as inclusion bodies and soluble aggregate in the E. coli protein expression system. However, the addition of 15% (v/v) glycerol to the protein purification buffer and the removal of N-terminal 24 amino acids of PmSTS helped to produce homogenous recombinant protein. Enzyme assay showed that recombinant PmSTS is active and specific to the C15 substrate FPP. The optimal temperature and pH for the recombinant PmSTS are 30 °C and pH 8.0, respectively. The GC-MS analysis further showed that PmSTS produces β-sesquiphellandrene as a major product and β-farnesene as a minor product. MSA analysis revealed that PmSTS adopts a modified conserved metal binding motif (NSE/DTE motif). Structural analysis suggests that PmSTS may binds to its substrate similarly to other plant sesquiterpene synthases.
DISCUSSION: The study has revealed that homogenous PmSTS protein can be obtained with the addition of glycerol in the protein buffer. The N-terminal truncation dramatically improved the homogeneity of PmSTS during protein purification, suggesting that the disordered N-terminal region may have caused the formation of soluble aggregate. We further show that the removal of the N-terminus disordered region of PmSTS does not affect the product specificity. The optimal temperature, optimal pH, Km and kcat values of PmSTS suggests that PmSTS shares similar enzyme characteristics with other plant sesquiterpene synthases. The discovery of an altered conserved metal binding motif in PmSTS through MSA analysis shows that the NSE/DTE motif commonly found in terpene synthases is able to accommodate certain level of plasticity to accept variant amino acids. Finally, the homology structure of PmSTS that allows good fitting of substrate analog into the catalytic active site suggests that PmSTS may adopt a sesquiterpene biosynthesis mechanism similar to other plant sesquiterpene synthases.